Novel drug-like RAR-Beta agonist induces BRCA1 to prevent neuropathic pain

Neuropathic pain (NP) is associated with profound gene expression alterations within the nociceptive system. DNA mechanisms, such as epigenetic remodelling and repair pathways have been implicated in NP. Here we have used a rat model of peripheral nerve injury to study the effect of a novel RARß-agonist, C286, currently under clinical research, in NP. A four week treatment initiated two days after the injury normalised pain sensation. Genome-wide and pathway enrichment analysis showed that multiple mechanisms persistently altered in the spinal cord were restored to preinjury levels by the agonist. Concomitant upregulation of DNA repair proteins, ATM and BRCA1, the latter being required for C286 mediated pain modulation, suggest that early DNA repair may be important to prevent phenotypic epigenetic imprints in NP. Thus, C286 is a promising drug candidate for neuropathic pain and DNA repair mechanisms may be useful therapeutic targets to explore. Overall design: Frozen micro dissected spinal cord samples were lysed with Teflon-glass homogenisers and QIAshredder columns (Qiagen). Total RNA was isolated with the Qiagen RNeasy® Mini kit. The RNA was assessed for purity and quantity using the Nanodrop 1000 spectrophotometer and assessed for quality on the Agilent Bioanalyzer 2100. Four hundred ng of intact high-quality total RNA (RIN>7.9) from each sample was then used as input to generate libraries for RNA-sequencing using the NEBNext Ultra II Directional kit (NEB, Cat.no: E7760S) following the manufacturer's recommendations. This protocol involved an initial step of mRNA selection using a poly-A isolation module (NEB, Cat.no: E7490) to select for mRNA with a mature polyA tail, followed by fragmentation prior to first cDNA synthesis and barcoding second-strand cDNA synthesised with indices for Illumina sequencing for final library amplification (10-cycles). The resulting libraries (342-421bp) were assessed on the Bioanalyzer 2100 for purity. The NEBNext Library Quant Kit for Illumina (NEB, Cat.no: E7630L) was used to calculate the quantity of each library. The quantification data was used to pool the libraries in equal molarity prior to performing a QC run on the MiSeq (MiSeq Reagent Kit v3 (150-cycle); Cat no: MS-102-3001). Further deep sequencing was performed on the pooled library over 2 lanes using a HiSeq4000 (by GENEWIZ) to generate roughly 26 million reads per sample.